Project Summary Addiction to pain medications, especially opiates, has become a major health problem and systems to guide the understanding of repeat overdose treatments are needed. Our proposal seeks to build overdose models for four drugs (fentanyl, methadone, codeine, and morphine) in a multi-organ system and evaluate the acute and repeat dose, or chronic effects, of overdose treatments such as Naloxone on overdose recovery, efficacy as well as off-target toxicity for cardiac, muscle, kidney and liver. We have developed a low cost system using human cells in a pumpless multi-organ platform that allows continuous recirculation of a blood surrogate for up to 28 days. This system emulates the distribution of a parental compound and the formation of metabolites among all ?organ? compartments and predicts potential toxicity and efficacy of drugs better than in vitro single human organ or animal models. We will develop two different overdose models for both male and female phenotypes based on nociceptors and B?tzinger Complex (B?tC) neurons as they contain -opioid receptors but are thought to have different roles in response to overdose and treatment. We will also integrate functional immune components in the UH3 Phase that has been demonstrated to enable organ specific or systemic monocyte actuation. In addition, models for cardiomyopathy and an infection model will be utilized to more accurately represent the effects of therapeutics on comorbidities. We will establish a PKPD in vitro model of overdose and treatment to enable prediction in clinical environments for a range of variables including age and drug-drug interactions. Once established the system could be used to evaluate novel pain therapeutics for efficacy and off-target toxicity as well as additional overdose treatments in future studies. Interconnected systems with continuous recirculation of a blood surrogate allows both the parent compound and its metabolites to be evaluated in the same system since it is a low volume platform. This interconnected system is better suited for preclinical drug testing than single organ systems for the same reason that human and animal models are currently the gold standards for toxicity and efficacy determination as they allow communication between the organ systems in the body. To construct a well defined system we will use a common serum free medium with microelectrode arrays and cantilever systems that are integrated on chip that allow for noninvasive electronic and mechanical readouts of organ function. UCF and Hesperos in collaboration with clinicians seek to radically change established practice in drug discovery by bypassing animal experiments and extensive clinical trials to provide treatments for diseases and clinical conditions such as overdose. We have already been working with regulatory authorities to prepare for eventual acceptance of the systems for regular use in INDs. Since Hesperos is already offering multi-organ evaluations as a service to the pharmaceutical industry and clinicians there is a direct translational element in the proposal.